Retroviral vectors for the treatment of tumors, and cell lines containing them

ABSTRACT

The invention relates to recombinant retroviral vectors, derived from Moloney MuLV, carrying a suicide gene susceptible of transforming an inactive substance into a toxic substance for cells going through a division process, said vectors being characterized by the presence in their structure of LTR sequences from variants of MuLV, and having the properties: (a) of not being inactivated during passage through the carcino-embryonic or line germinal cells of mice; (b) the expression of the suicide gene kills only the cells in the course of division.

This application claims priority on and is the National Stageapplication of PCT International Application No. PCT/FR93/01259 filed on16 Dec. 1993.

Gene therapy offers novel possibilities for the treatment of tumors.

Among the difference options, the objective of gene transfer is to killthe tumor cells either indirectly by the induction or the reinforcementof an immune response of the host (S. A. Rosenberg, Cancer Res. 51:5074(1991) or directly by the insertion of a "suicide gene" (F. L.Moolten, Cancer Res. 46: 52-76 (1986)). The most studied suicide gene isthat coding for thymidine kinase of type 1 herpes simplex virus(TK-HSV1) (G. B. Elion et al., J. Antimicrob. Chemother. 12: 9-17(1983)). This enzyme phosphorylates nucleoside analogues such asganciclovir (GCV). The monophosphate molecules are then converted intothe triphosphate forms by cellular enzymes. The GCV triphosphate(GCV-TP) can then be incorporated into the DNA during cell division,blocking elongation and thus leading to the death of the cell. GCV-TP isthus only toxic for dividing cells.

This approach is particularly interesting for the treatment of tumorswhich are constituted of rapidly dividing cells in a tissue constitutedof non-proliferating cells, and the expression of TK-HSV1 by the cellsof a tumor situated within an organ whose cells are not dividing oughtto permit the specific destruction of these cells. Furthermore, it ispossible to target gene transfer into these tumor cells by using for thetransduction of TK-HSV1 recombinant retroviruses whose genome can onlybe integrated and expressed in dividing cells.

The first experimental ablation model by GCV of tumor cells expressingTK-HSV1 was set up by Moolten (F. L. MOOLTEN, Cancer Res., 46, 1986, p.5276-5281); F. L. MOOLTEN and J. M. WELLS, J. Natl. Cancer Inst., 82,1990, p. 297-300). He showed an antitumor effect of GCV on the growth oftumor cells in mice after transfection of the TK-HSV1 gene in vitro, andreimplantation in the animal. Recently, the elimination of microscopicexperimental cerebral tumors by stereotaxic injection of cells producingTK-HSV1 retroviruses and treatment by GCV has been reported by Culver(K. W. Culver et al., Science 256: 1550-1552 (1992)).

However, in this model the authors were unable to analze the effect ofsuch a treatment on macroscopic established tumors which rapidly becomelethal. Now, in patients solid tumors are the principal target of thistype of treatment and the problem of the transduction of the suicidegenese in the context of a tumoral mass is much more problematical.

In man, hepatic metastases are a frequent complication of digestivecancers. Partial hepatectomy is only possible in about 15% of cases, andother treatments such as local chemotherapy or immunotherapy havehitherto given only modest, even disappointing results. The present workshows the efficacy of the treatment of experimental hepatic metastasesin the rat after in vivo transfer of the TK-HSV1 gene by directinjection of murine fibroblasts producing recombinant retroviralparticles.

In this context, one of the objectives of the present invention is thedevelopment of a packaging cell-recombinant retroviral vector systemutilizable in therapy to treat established tumors, in particular hepatictumors, and in which the expression product of the recombinant gene iscapable of converting a non-toxic prodrug into a drug toxic for thecells expressing said recombinant gene.

More precisely, the objective of the present invention is to construct arecombinant retroviral vector capable, after transformation of afibroblast packaging line, of producing retroviral pseudoparticleswhich, after infection of dividing cells, results in the expression ofthe transduced gene without leading to inactivation of said vectorswhich occurs and has been described in particular with MuLV-Moloney.

The murine transgenic lines of the Mov series were established byJaenisch et al. by infection of mouse embryos with the Moloney-MuLVvirus before implantation (Jaenisch et al., Cell 24: 519-529 (1981)).

At this stage of development the animals are usually resistant to theinfection by the MuLV, the hypothesis being that the proviral DNAundergoes a process of methylation acquired during successive celldivisions and this methylation prevents the transcription of the DNA.

However, with some Mov lines, the MuLV genome may be reactivated at aparticular stage of development and the rodents develop leukemiasinduced by this oncogenic virus. This is the case for the followingthree lines: Mov3, Mov9 and Mov13.

This phenotype might be due to mutations in the LTR.

IN THE FIGURES

FIG. 1 depicts the structure of the recombinant retroviral vector pMTK.

FIG. 2 shows the results of experiments on in situ administration into ahepatic tumor of transfected lines carrying a Tk-HSV1 gene followed bytreatment with a nucleoside analogue.

Hence, the object of the present invention is the construction of arecombinant retroviral vector derived from the MuLV-Moloney carrier of asuicide gene capable of converting an inactive substance into asubstance toxic for dividing cells, and characterized in its structureby the presence of LTR sequences derived from such variants of the MuLVwhich have the property of not being inactivated during passage throughmouse carcino-embryonic or germ line cells and capable of transfectingcells which divide, in particular tumor cells.

The suicide gene used is the thymidine kinase gene of the herpes simplexvirus.

The invention also relates to packaging lines transfected by thisrecombinant vector derived from the Moloney retrovirus and comprisingthe TK-HSV1.

The invention relates more particularly to the M11 line deposited withthe C.N.C.M. on 15 Dec. 1992 under the number No. I-1278.

The in situ administration into a hepatic tumor of transfected linescarrying a TK-HSV1 gene followed by a treatment by a nucleoside analoguesuch as ganciclovir leads to a reduction of the tumor mass by more than90% under the experimental conditions described below (cf. FIG. 2).

Finally, the invention relates to therapeutic compositions containingthe cell line transfected by recombinant retroviruses, utilizable incombination with a prodrug to lead to selective destruction of the tumorcells.

The invention takes advantage of three separate phenomena:

1) the injection of the composition into dividing cells, themselvessurrounded by quiescent cells;

2) the presence in this composition of recombinant retroviral vectorswhich only infect dividing cells, and consequently the gene is expressedonly in these cells;

3) the expression of the suicide gene only kills dividing cells. p Inother words, all of the dividing cells infected by the recombinantretroviruses, and expressing the TK-HSV1 gene are capable of beingkilled after treatment with the nucleoside analogue which may thus beconverted into a nucleoside triphosphate and stops the elongation of theDNA after its incorporation into the growing DNA chain.

The vulnerable cells in this phenomenon are thus

the target tumor cells; and

the transfected fibroblasts of the composition of the invention.

The objective of the experimental conditions given below is toillustrate the construction of the retroviral vector which makespossible the selectivity of the "killer" effect described above, thepreparation of the lines transfected by such a vector and thetherapeutic efficacy of the composition containing these lines when thetreatment is combined with the administration of a prodrug such asganciclovir.

1) Construction of the retroviral vector pMTK

The construction of a vector is described in which the TK-HSV1 gene hasbeen placed under the control of a 5' LTR of the mutant MuLV strainsMov3, Mov13 and Mov9.

The use of this plasmid for the transfection of CRIP fibroblast cells isdescribed subsequently.

The ligations of the different restriction fragments of the strainsMov3, Mov13 and Mov9 were carried out in the following manner:

5'-LTR

Mov3 up to the BamHI site (-350 from the transcription start).

Mov13 from the BamHI site (-350) up to the KpnI site (+30).

Mov9 from the KpnI site (+30) up to the PstI site (+560) (contains thepackaging sequence).

The non-coding sequence at the 3' of the ClaI site (+7674) up to U3)(+7817 is derived from Mov3.

3'-LTR

Mov3 up to the BamHI site situated at position 7910 (or -350).

Mov13 up to the end of U5.

The 1100 base pairs fragment containing the TK-HSV1 gene and defined bythe SalI and NotI ends was inserted into the polyliner at the sitesindicated in FIG. 1.

All of these cloning operations were carried out using standardprocedures (Maniatis et al. (1982), Molecular Cloning, a laboratorymanual).

FIG. 1 presents the recombinant retroviral vector.

2) Cell cultures and transfection, establishment of the packaging lineM11:

The Ψ CRIP fibroblast cells (O. DANOS et al., Proc. Natl. Acad. Sci.USA, (1988), 85: 6460-64) were cotransfected with the retroviral vectorpMTK (20 μg) expressing TK-HSV1 under the control of the LTR and aselection plasmid (1 μg) (pWLNeo, Stratagene). Several clones wereisolated by G418 selection. The production of viral particles by theseclones were then tested by infection of TK-L cells and selection ofcells on HAT. The infection was carried out with serial dilutions of thesupernatant containing the viral particles, and counting the number ofcolonies obtained under these conditions enables the infectious titer tobe determined. The selected clone M11 possesses a titer of 5×10⁵ viralparticles/ml. The line NB16 (N. FERRY et al., Proc. Natl. Acad. Sci.(1991) 88: 8377-81), also derived from Ψ CRIP cells, was used toevaluate the infection of the tumor cells. It produces viral particlesexpressing the nls-LacZ gene with with an infectious titer of 10⁴ viralparticles/ml.

The DHDK12 line is a cell line established from chemically induced coloncancer in the rate BDIX described in Martin et al., Virchows Archiv. A.Pathol. Anat. (1991) 418: 193.

The invention also relates to the populations of fibroblast cellstransformed by a recombinant retroviral vector carrying the suicidegene.

3) Therapeutic protocol

Syngenic male BDIX rats were used. An isolated liver tumor was inducedby direct injection of the DHDK12 cells under the capsule of the liver.On day 5 the liver tumors had a diameter included between 2 and 3 mm andcontained approximately 150 million cells.

At this time, the rats received an intra-tumoral injection of thefibroblasts producing recombinant retroviruses (20×10⁶ cells).

The animals of the control group received NB16 fibroblast cells whichproduced a recombinant retrovirus expressing the nls-lacZ gene. Thisgene codes for a beta-galactosidase with a localization signal in thenucleus and can thus serve as an indicator gene to monitor the infectionof the tumor cells in vivo. The NB16 clone produces the virus with atiter of 10⁴ plaque-forming units per milliliter. The group of treatedanimals were infected with the M11 line which produced 5×10⁵ infectiousparticles per milliliter.

After the intra-tumoral injection of the packaging cells, the animalswere left for a period of 5 days in order that the infection of thetumor cells by the recombinant retrovirus may occur.

Five days later, i.e., on day 10, all the rats received ganciclovir(GCV) (Syntex) for five days at a dose of 150 mg/kg twice a day by theintraperitoneal route. The rats were then sacrificed at the end of thetreatment with ganciclovir, i.e., 15 days after the first injection andan autopsy was performed comprising the measurement of the size of thetumors and a pathological anatomy study.

FIG. 2 summarizes the experimental protocol described above and theresults obtained on the reduction of the tumors described below.

RESULTS

Control group (number of rats: 12):

On autopsy, all of the injected lobes showed macroscopic tumors, themean maximal diameter of which was 6.5±2.1 mm (extremes: 3 and 10 mm).Their volume is calculated according to the formula V=A×B² /2, in whichA is the maximal diameter, and B is the smallest diameter measured atthe largest cross-section of the tumor (G. Carlsson et al., 1983), J.Cancer Res. Clin. Oncol. 105: 20). The volume calculated under theseconditions was 86.3±65.1 mm³ (mean±S.D.).

The pathological examination of the tumors revealed a typical appearanceof a poorly differentiated adenocarcinoma, consisting essentially oftumor cells organized in lobules of variable thicknesses. The stromaappeared fibrotic and surrounded with mononuclear inflammatory cells.The percentage of tumor cells in the tumor was estimated at 60%.

The nuclear beta-galactosidase activity is detected in Xdale-stainedcryostatic sections in the 6 control livers analyzed. The fraction ofinfected tumor cells was less than 1%.

Treated group (number of rats: 13):

The mean diameter of the tumors was 3±1.1 millimeters (extremes: 0.5 and4 mm) and the volume was 8.1±6.7 mm³ (P<0.0001, Mann and Whitney test)(FIG. 2). The pathological examination of the tumors revealed theefficacy of this treatment by a very considerable dimunution in the sizeof the tumors. Of the 11 livers analyzed (2 were used for the lacZanalytical control), two had tumors showing a reduction of 60 to 20% ofthe tumor cells. These cells were poorly organized and necrotic. Theywere grouped together in a fibrotic reaction surrounding inflammatorymononuclear cells and a cancerous hyperplasia. In 5 other livers lessthan 10% of cancer cells could be detected, with a massive fibroticreaction. In the last four livers, a few rare cancer cells were seen anda massive necrosis of the tumor surrounded by a fibrotic reaction wasobserved.

In all of these rats, there were signs of a parenchymatous regeneration.

These studies show a regression of established tumors after in vivotransfer of a suicide gene.

In patients a precise targetting of the injection of the transformedpackaging fibroblasts into the tumor mass can be facilitated byultrasonic assistance or with the aid of any microprobe whose functionis the localization of said tumoral masses.

Knowing that the level of infected cells expressing the TK-HSV1 gene wasless than 10% and that the tumor is reduced by more than 90% afteraddition of ganciclovir, it may be supposed that the phenomenon of"metabolic cooperation", already described by Moolten (Moolten, 1986) orthe "proximity effect" (Culver, 1992) is also effective in the case of alarge tumor mass. This phenomenon could be explained by the transfer ofthe phosphorylated ganciclovir from the cells expressing the TK-HSV1gene to the cells not expressing it.

The use of the packaging fibroblast cells, transformed by a recombinantretroviral vector carrying a suicide gene as medicine combined with atreatment with ganciclovir seems to be efficacious for the reduction oftumoral masses and the treatment of established cancers.

The invention is not limited to the embodiment which has beenillustrated above, in particular to the use of the TK-HSV1/ganciclovircouple. The specialist skilled in the art is able to imagine otheractive couples which may be used for the same purpose. As a killer genemention should be made, for example, of the thymidine kinase gene of thecytomegalovirus coupled to ganciclovir. The gene sequence and thestructure of the protein of this thymidine kinase show that the latteris quite different from the cellular kinases or the kinase of the herpessimplex 1 virus. Mention may also be made of a bacterial enzyme,cytosine deaminase, which is capable of converting 5-fluorocytidine intoa toxic nucleotide analogue 5-fluorouracil. Finally it is possible toimage any other system of condition toxicity based on an enzyme-drugcouple and which, as a result of the system described above, enablesdividing cells to be killed specifically.

We claim:
 1. A genetically altered Moloney MuLV retroviral vectorcomprising:(a) a suicide gene comprising the herpes simplex virusthymidine kinase gene (HSV-tK); and (b) Mov3, Mov9 and Mov13 LTRsequences;wherein said genetically altered Moloney MuLV retroviralvector is not inactivated by passage through mouse carcinoembryonic orgerm cell lines.
 2. A fibroblast packaging cell line transformed withthe retroviral vector of claim
 1. 3. A method for killing solid tumorcells, said method comprising the steps of:(a) injecting into a solidtumor a fibroblast packaging cell line transformed with a geneticallyaltered Moloney MuLV retroviral vector comprising:(i) the herpes simplexvirus thymidine kinase gene (HSV-tK); (ii) Mov3, Mov9 and Mov13 LTRsequences; wherein said genetically altered Moloney MuLV retroviralvector is not inactivated by passage through mouse carcinoembryonic orgerm cell lines; and thus transferring the retroviral vector whichexpresses the HSV-tK gene into the tumor cells; and (b) killing thetumor cells in said solid tumor by administering to the solid tumor aprodrug that is converted by the thymidine kinase expressed from theHSV-tK gene of step (a), said prodrug is converted by said thymidinekinase to a toxic product that kills the tumor cells.